DESCRIPTION (provided by investigator): The pathogenic protist Entamoeba histolytica is estimated to be responsible for 50,000 to 100,000 deaths each year and is a significant source of morbidity and mortality due to parasitic disease in humans, particularly in developing countries. E. histolytica has a simple life cycle, existing in either an infectious cyst form or as motile trophozoites which can reside in the anaerobic confines of the human colon. Although amoebiasis is currently treated with metronidazole, development of additional agents effective against E. histolytica infection is warranted. In order to identify suitable targets, it is importat to first develop a more in- depth understanding of the metabolism and physiology of this pathogen. The amitochondriate E. histolytica relies on an unusual PPi-dependent glycolytic pathway as a primary means for ATP generation. Our long-term goal is to provide a better understanding of E. histolytica metabolism as this unique eukaryotic pathogen traverses the different environments encountered during various stages of infection. The objective of this proposal is to investigate the roles of acetate kinase (ACK) and ADP-forming acetyl-CoA synthetase (ACD), two enzymes that may play key roles in the PPi-dependent glycolytic pathway. ACK is primarily a bacterial enzyme that catalyzes the conversion of acetyl phosphate + ADP to acetate + ATP in a fully reversible reaction. The Entamoeba enzyme is unique in that it catalyzes the conversion of acetyl phosphate + Pi to acetate + PPi, and thus may function to supply PPi for glycolysis. Preliminary results have shown that ACK is essential, consistent with this critical role. ACD catalyzes the interconversion of acetate and acetyl-CoA and is present in only a handful of eukaryotic microbes including the pathogens Plasmodium. If used to break down acetyl-CoA produced from glycolysis, ACD's effect would be to increase ATP production. However, ACD may instead play a role in utilizing acetate produced by the microbiota in the large intestine. Specific Aim 1 is to identify active site residues in ACK and ACD responsible for substrate binding and catalysis. Amino acid residues in each enzyme will targeted for alteration based on structural analysis and analogy to closely related enzymes. The enzyme variants will be analyzed to determine the effects of these alterations on substrate binding and catalysis. Specific Aim 2 is to determine the physiological roles of ACK and ACD in E. histolytica. Antisense RNA-mediated gene silencing and a novel RNAi rescue method being developed for examination of essential genes will be used to examine the roles of ACK and ACD under different growth conditions. Metabolic profiling will be used to determine the metabolic effects of the different growth conditions on the wild type and RNAi/RNAi rescue mutants. The results of these studies will fill a gap in our knowledge of Entamoeba metabolism. These enzymes in their proposed roles would play an important role in growth and survival in the human host as Entamoeba traverses the intestinal tract during infection, but such roles need to be confirmed.